Abstract

4-Methylaminorex, a potential psychostimulant drug of abuse, exists as four stereoisomers: cis-4R,5S, cis-4S,5R, trans-4S,5S, and trans-4R,5R, which were shown previously to possess stereospecific effects. This study characterized their pharmacokinetic and tissue distribution profiles, and metabolic turnover to norephedrine and norpseudoephedrine, in male Wistar rats. The rats received each isomer intravenously, intraperitoneally, or orally, followed by blood sample collection via cannula (pharmacokinetic study), or tissue sample collection at predetermined time points (tissue distribution study). The samples were analyzed for cis- and trans-isomers, and when appropriate for norephedrine and norpseudoephedrine, with gas chromatography/mass spectrometry. Trans-4S,5S-, cis-4R,5S-, and cis-4S,5R-isomers behaved comparably kinetically (volume of distribution 1.7-2.3 l/kg, distribution half-life 3.8-7.0 min, elimination half-life 35-42 min, and bioavailability 32-57% intraperitoneally or 4-16% orally), whereas trans-4R,5R-isomer differed from the others, with a longer elimination half-life (118-169 min) and higher bioavailability (100% intraperitoneally or 83% orally). The highest isomer concentrations were observed in the kidney followed most frequently by the liver, brain, muscle, and last by fat and blood. The elimination half-lives of the stereoisomers from the tissues were generally similar to those in blood. No pharmacologically significant amounts of norephedrine or norpseudoephedrine were detected in blood or the brain. In conclusion, differences between the stereoisomers of 4-methylaminorex in the pharmacokinetics and tissue distribution are described. However, these differences are not compatible with, and thus may not account for, the distinct behavioral and neurochemical effects of the stereoisomers demonstrated previously. Furthermore, metabolic turnover to norephedrine and norpseudoephedrine does not seem to contribute significantly to 4-methylaminorex pharmacology.

The phenylisopropylamine derivative 4-methylaminorex (2-amino-4-methyl-5-phenyl-Δ2-oxazoline) is a sympathomimetic agent that has been found on the clandestine market with street names such as “U4Euh” and “Ice” (Davis and Brewster, 1988; Klein et al., 1989; Gaine et al., 2000). The drug has been misrepresented by dealers as cocaine or methamphetamine (Davis and Brewster, 1988), and anecdotal evidence describes it as inducing stimulant-like effects such as euphoria, vigor, restlessness, tremor, tachycardia, and increase in blood pressure. Also an overdose fatality has been attributed to 4-methylaminorex abuse (Davis and Brewster, 1988). More recently, Gaine et al. (2000) describe a case series of patients suffering from pulmonary hypertension, a disease with poor prognosis, caused by 4-methylaminorex. Although the current extent of 4-methylaminorex abuse is not known, users' recent experiences, opinions, and recommendations regarding its effects, dosage, intake manners, and synthesis methods can be found on drug-culture related Web sites (e.g., http://www.erowid.org or https://www.thehive.ws), which illustrates that the drug is a subject of ongoing interest, and further highlights its abuse potential.

Given that the effects of individual stereoisomers are clearly distinct, the question also arises whether there are differences in the pharmacokinetics of the isomers. There are, however, only limited data available on the kinetic and metabolic properties of the stereoisomers of 4-methylamin- orex. Somewhat surprisingly, our previous studies show that the concentrations of the least effective isomer trans-4R,5R might be higher than those of the other isomers in rat brain dialysate or tissue samples at given time points (Kankaanpää et al., 2001; Kankaanpää et al., 2002). The published data on the metabolism of 4-methylaminorex are limited to a single study. Henderson et al. (1995) showed that a mixture containing mainly the cis-isomers was excreted in rat urine much as unchanged drug (60% of total excretion) with three metabolites present: two pharmacologically poorly characterized oxazoline-derivatives and the synthetic precursor norephedrine. It is not known whether norpseudoephedrine or other metabolites are formed from the trans-isomers.

In general, the pharmacokinetics of a drug is essential in comprehensive understanding of the characteristics of its abuse. Harmful patterns of consumption can be predicted by means of pharmacokinetic parameters such as bioavailability, volume of distribution, and half-life (Quinn et al., 1997). The stereospecific pharmacokinetics of a drug also has an impact on analyses and evaluations of clinical and forensic interest, and in particular, it may account for the drug's stereospecific actions. Consequently, the aim of this study was to characterize the stereospecific pharmacokinetics and tissue distribution patterns of the isomers of 4-methylaminorex, a potential psychostimulant drug of abuse. Pharmacokinetic data were used to evaluate kinetic differences between the isomers and to understand the time course of blood concentrations in the overall pharmacological effects of the isomers. This was supplemented with a tissue distribution study to gain further insight into kinetic behavior and its differences between the isomers. Finally, concentrations of norephedrine and norpseudoephedrine in blood and the brain were measured to evaluate their contribution to 4-methylaminorex pharmacology.

Materials and Methods

Drugs and Chemicals. The four optical stereoisomers (trans-4R,5R, trans-4S,5S, cis-4R,5S, and cis-4S,5R) of 4-methylaminorex were prepared at the Laboratory of Organic Chemistry (University of Helsinki, Helsinki, Finland) using the synthesis methods described previously (Poos et al., 1963; Klein et al., 1989). The identity of the isomers was confirmed by determining their melting points and rotation angles, and the 1H NMR and 13C NMR spectra. For animal experiments, the isomers were dissolved in a small volume of 2 M HCl, the pH was adjusted to physiological level with 2 M NaOH, and the solution was then brought to volume with purified water. The amounts of HCl and NaOH were calculated so that the final drug solution was roughly isotonic physiologically. Doses were calculated as free base, and the drugs were administered at a volume of 1 ml/kg (intravenous and intraperitoneal injections) or 5 ml/kg (oral administration).

Norpseudoephedrine hydrochloride was donated by the National Institute on Drug Abuse (National Institute on Drug Abuse, Bethesda, MD) and norephedrine hydrochloride by the Orion Corporation (Espoo, Finland). Methylmexiletine and carbamazepine were obtained from Boehringer Ingelheim GmbH (Ingelheim, Germany), and Geigy (Basel, Switzerland), respectively. N-(tert-Butyldimethylsilyl)-N-methyltrifluoroacetamide (N-methyl-N-t-butyldimethylsilyl trifluoroacetamide) was purchased from Aldrich Chemical Co. (Milwaukee, WI), and heptafluorobutyric anhydride was from Fluka (Buchs, Switzerland). The other common reagents used were of the highest quality.

Animals. Adult male Han:Wistar rats, weighing 250 to 350 g (pharmacokinetic study) or 300 to 400 g (tissue distribution study), were used. The rats were obtained from Harlan Nederland B.V. (Horst, The Netherlands), at least 1 week before the experiments, and they were housed in a temperature-controlled room (22 ± 2°C) with a light cycle of 12 h. The lights were on from 6:00 AM to 6:00 PM, during which time all the experiments were conducted. The animals had free access to standard laboratory chow (Altromin Nr. 1314; Chr. Petersen A/S, Ringsted, Denmark) and tap water, unless otherwise stated. The local Institutional Animals Care and Use Committee, and the chief veterinarian of the county administrative board approved the experiments, which were conducted according to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes.

Pharmacokinetic Experiments. At least 6 days before the experiments, the rats underwent surgery for the implantation of vascular cannulas. During the surgery, the rats were anesthetized using halothane gas (Halothane Liquid; Rhodia Ltd., Bristol, UK). The right jugular vein was exposed and cannulated with a heparinized (Heparin Leo 5000 IU/ml; Leo Pharma A/S, Ballerup, Denmark, diluted with saline to 31 IU/ml) polyethylene-50 tube for blood sampling. The tip of the cannula was advanced to the right atrium of the heart. When intravenous administration so required, the left femoral vein was also exposed and cannulated with a heparinized polyethylene-10 tube, which was advanced to the inferior vena cava. With other routes of administration, the femoral vein was left uncannulated. The cannulas were then passed under the skin and fixed near the base of the neck. After surgery, the rats received a subcutaneous injection of buprenorphine (0.05 mg/kg; Temgesic 0.3 mg/ml; Reckitt and Colman Ltd., Hull, UK), and a intravenous bolus of a mixture of sulfadiazine and trimethoprime (50 and 10 mg/kg, respectively; Tribrissen mite 200 mg/ml and 40 mg/ml; Schering-Plough A/S, Farum, Denmark, diluted with saline to 50 and 10 mg/ml). The antibiotic mixture was also applied to the wounds. The cannulas were flushed with heparinized saline every other day.

On the test day, the rats received intravenously, orally, or intraperitoneally a single bolus of one isomer at the dose of 2 mg/kg. This dose was chosen because some animals had died after intravenous administration of trans-4S,5S-isomer at higher doses in preliminary experiments. The intravenous injection was given slowly (30 s) via the femoral vein-cannula. For oral administration, an oral gavage with a syringe was used in rats that had fasted for at least 4 h. The intraperitoneal injection was given into the left lower quadrant of the abdomen. Blood samples (150 μl) were drawn into a heparinized syringe via the jugular vein-cannula as follows: 0 (2-10 min before drug administration), 2, 10, 20, 30, 40, 60, 80, 150, 180, 210, and 240 min after the drug administration. The collected blood volume was replaced with 150 μl of saline after each sample. The blood samples were stored in vials at 5°C and analyzed for 4-methylaminorex concentrations within 1 to 5 days. According to our preliminary experiments, blood sample concentrations would remain sufficiently stable during this period of storage. When measured in the same sample at 1-week interval, the relative isomer concentrations on day 7 were as follows (as percentage of day 1 concentrations ± 95% probability limits; n = 4-6): 94 ± 11% for cis-4R,5S, 109 ± 9% for cis-4S,5R, 107 ± 15% for trans-4S,5S, and 106 ± 11% for trans-4R,5R.

Tissue Distribution Experiments. On the test day, the rats received a single bolus of one isomer at the dose of 5 mg/kg intraperitoneally, into left lower quadrant of the abdomen. At time points of 30, 60, 100, 150, 210, or 280 min after the injection, the rats were lightly sedated with carbon dioxide and decapitated. In our preliminary tests, this light exposure to carbon dioxide did not affect the 4-methylaminorex concentrations in tissues. Immediately after the decapitation, trunk blood was collected in a heparinized tube. The brain, liver, left kidney, and samples of pectoral muscle and subcutaneous fat were then dissected out and placed in vials. The blood and tissue samples were stored at 5°C until analyzed within a few days.

Chemical Analyses. The concentrations of the stereoisomers of 4-methylaminorex were determined from blood and the tissues with gas chromatography/mass spectrometry (GC/MS). In addition, blood and brain tissue samples were analyzed for the presence of the 4-methylaminorex active metabolites norephedrine and norpseudoephedrine.

Tissue samples required pretreatment before extraction. They were weighed and homogenized in a 4-fold quantity of freshly made 0.1 M HClO4. After centrifugation, the supernatants of brain tissue and fat samples were washed with 5 ml of n-hexane, whereas the other tissues needed no further processing before extraction.

The quantitative determination of cis- and trans-4-methylaminorex concentrations in blood and the tissue samples was performed essentially as described previously (Kankaanpää et al., 2001). The method used distinguishes the cis- and trans-isomers by their retention times, but not the two cis-isomers from each other, or the two trans-isomers from each other. Briefly, a 100-μl (pharmacokinetic study) or 1-ml (tissue distribution study) aliquot of blood, or a 1-ml aliquot of pretreated tissue sample was mixed with 1 ml of 0.5 M NaOH and 5 ml of toluene containing carbamazepine (8 μg/100 ml) as an internal standard. After centrifugation, the toluene layer was evaporated to dryness, and the derivatization reagent, 120 μl of N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide/acetonitrile (1:6), was added to the residue. After a 30-min incubation at 80°C, a 1-μl aliquot of the mixture was injected into the GC/MS apparatus consisting of an Agilent 5890 Series GC system and Agilent 5973 network mass selective detector (Agilent Technologies, Palo Alto, CA). The system was operated in the splitless injector mode. The GC column was a DB-35MS, 30 m in length, with an internal diameter of 0.32 mm and a film thickness of 0.25 μm (J&W Scientific Inc., Folsom, CA). Helium was used as the carrier gas. The inlet and detector temperatures were maintained at 250 and 280°C, respectively. The column temperature was initially 150°C with a hold time of 2.0 min and was increased 15°C/min to 320°C, with a final hold time of 3.0 min.

The extraction recovery was determined from spiked tissue samples as follows: an aqueous solution of 4-methylaminorex (cis-4S,5R; 0.010 mg/ml; 50 μl) was injected into several loci in the intact tissue sample with a hypodermic needle. After overnight incubation at 5°C, the tissue samples were pretreated and extracted as described above. The blood samples were spiked and extracted as usual. The extraction recovery (n = 7-10) from blood, brain, liver, and kidney ranged from 77 to 81% compared with plasma samples, whereas the corresponding values for muscle and fat were 89 and 107%, respectively. Based on these results, it was concluded that standard curves constructed from spiked rat plasma samples could be used to determine 4-methylaminorex in all the matrices studied, with tissue-specific correction made according to the extraction recovery. The rest of the validation data correspond to that presented earlier (Kankaanpää et al., 2001), except that the limit of quantification was set at 0.020 μg/ml.

Norephedrine and norpseudoephedrine were extracted by mixing 1 ml of the sample (blood or pretreated brain tissue) with 1 ml of 0.5 M NaOH and 5 ml of toluene, with methylmexiletine as the internal standard (0.500 μg/sample). After centrifugation and separation of the phases, the derivatization reagent, 8 μl of heptafluorobutyric anhydride per sample, was added to the toluene layer, vortexed, and washed with 1 ml of saturated NaHCO3. The mixture was then centrifuged and the toluene layer evaporated to dryness, after which the dry residue was dissolved in 100 μl of toluene and injected into the GC/MS apparatus at a volume of 3 μl. The apparatus and the analysis conditions were comparable with those described above, except that the GC column was a DB-5MS, 30 m in length, with an internal diameter of 0.32 mm and a film thickness of 1 μm (J&W Scientific Inc.). The limit of quantification was set at 0.010 μg/ml. Relative standard deviation was 4.8% at a concentration level of 0.050 μg/ml and 8.5% at the limit of quantification.

Pharmacokinetic and Statistical Analyses. The data from the pharmacokinetic study were analyzed using model-independent methods. When appropriate, compartmental analyses were also used to obtain rate constants for absorption, distribution, and elimination (kab, kdi, and kel, respectively). The data were fitted using the nonlinear least-squares fitting method of the Systat version 10 program (SPSS Science, Chicago, IL). The goodness of fit and the most appropriate model were determined by assessing the randomness of the scatter of actual data points around the fitted function and by using Akaike's information criterion (Akaike, 1976). The intravenous and intraperitoneal data were best described with a two-compartmental model and a one-compartmental model with the absorption phase, respectively. No model could be fitted with the oral data of the isomers, with the exception for trans-4R,5R. The peak concentration (Cmax) and time to reach it (Tmax) were taken directly from the data. The area under the curve from 0 to 240 min (AUC0-240) or to infinity (AUC0-∞), and the area under the first moment curve from 0 min to infinity (AUMC0-∞) were calculated using the trapezoidal method. The terminal area from the last sampling point to infinity was extrapolated using the kel value. The mean residence time (MRT), bioavailability (Fbioav; using the average AUC0-240 of the intravenous data as the reference value), total clearance (CL), volume of distribution at steady state (Vdss), absorption half-life (T1/2ab), distribution half-life (T1/2di), and elimination half-life (T1/2el) were calculated using standard formulas (Rowland and Tozer, 1995).

In the tissue distribution study, pharmacokinetic parameters for blood and the tissues were calculated with average 4-methylaminorex concentrations at each time point. The tissue-specific rate constant k for the declining phase was calculated using the nonlinear least-squares fitting method as described above. The best fit in all the tissues was obtained with the weighting factor 1/(observed concentration)2. T1/2 and AUC30-150 were likewise calculated as described above.

Statistical analyses were performed using the one-way ANOVA or Kruskel-Wallis tests. One-way ANOVA was followed by Bonferroni's test, adjusted for the appropriate number of comparisons. The two-sample t test, Mann-Whitney U test, and Pearson correlation were calculated when appropriate. Data are expressed as mean ± S.E.M. or median (minimum-maximum).

Results

Pharmacokinetics of the 4-Methylaminorex Isomers.

The blood concentration-time profiles of the stereoisomers of 4-methylaminorex after intravenous, intraperitoneal, and oral routes of administration are illustrated in Fig. 1. The pharmacokinetic parameters calculated from the data are summarized in Table 1.

Blood concentration-time profiles of the stereoisomers of 4-methylaminorex after intravenous, intraperitoneal, and oral routes of administration at the dose of 2 mg/kg (n = 3-6). Error bars covered by symbol are not shown.

Pharmacokinetic parameters of the stereoisomers of 4-methylaminorex after intravenous, intraperitoneal, or oral administration at the dose of 2 mg/kg

The data are presented as mean ± S.E.M. or median (min-max). kab, kdi, and kel, absorption, distribution, and elimination rate constants; T½ab, T½di, and T½el, absorption, distribution, and elimination half-lives; MRT, mean residence time; AUC0-240 and AUC0-∞, area under the curve from 0 min to 240 min or infinity; AUMC0-∞, area under the first momentary curve from 0 min to infinity; Cmax and Tmax, observed maximum concentration and time to reach it; Fbioav, bioavailability.

The kinetic behavior of trans-4S,5S-, cis-4R,5S-, and cis-4S,5R-isomers in blood was virtually the same, whereas that of trans-4R,5R-isomer differed markedly from the others. The most marked differences were observed in the elimination kinetics. The elimination of trans-4R,5R-isomer was approximately 3 times slower than that of the others (T1/2el 119-129 versus 35-42 min; MRT 162 versus 41-52 min). It needs to be emphasized that although the most optimal sampling period for trans-4R,5R-isomer may had been longer than the used 240 min, appropriate and valid fit was obtained with all the isomers. One-way ANOVA showed statistical differences between the isomers in the following parameters: CL [F(3.17) = 5.0; p = 0.011 (intravenous data)], kel [F(3.17) = 12.7; p < 0.001 (intravenous data) and F(3.11) = 3.9; p = 0.040 (intraperitoneal data)], T1/2el [F(3.17) = 41.8; p < 0.001 (intravenous data) and F(3.11) = 15.1; p < 0.001 (intraperitoneal data)], and MRT [F(3.17) = 60.4; p < 0.001 (intravenous data)]. According to post hoc comparisons, most often trans-4R,5R-isomer differed from all the others. The elimination kinetics remained the same, regardless of whether the isomers were administered intravenously or intraperitoneally.

The blood and tissue concentrations of the stereoisomers of 4-methylaminorex after intraperitoneal administration are illustrated in Fig. 2. The tissue-specific pharmacokinetic parameters are presented in Table 2.

Concentration-time profiles of the stereoisomers of 4-methylaminorex in blood, and brain, fat, muscle, liver, and kidney tissues after intraperitoneal administration at the dose of 5 mg/kg (n = 2-3). Error bars covered by symbol are not shown.

Pharmacokinetic parameters of the stereoisomers of 4-methylaminorex in blood and tissues after intraperitoneal administration at the dose of 5 mg/kg

The data are presented as means; no S.E.M. is shown because the data are calculated from the average concentrations. AUC30-150 area under the curve from 30 min to 150 min; T½ elimination half-life.

Analogously with the blood concentrations, the tissue concentrations of the trans-4R,5R-isomer were markedly higher than those of the other isomers, which in turn were mostly similar to each other. The highest concentrations were found typically in the kidney followed by the liver, brain, and muscle. The lowest concentrations were in the fat and blood. One exception was the liver concentration of cis-4R,5S-isomer, which was lower than the brain concentration of the same isomer, and at least 4-fold lower than the liver concentrations of the other isomers. Penetrability into the tissues, expressed as AUC30-150tissue/AUC30-150blood ratio, was almost identical between the isomers, although the ratios for the trans-4R,5R-isomer seemed to be somewhat lower. As suggested by the low liver concentration, the penetrability of the cis-4R,5S-isomer into this organ was lower than that of the other isomers. The elimination rate of the isomers from the tissues, expressed as T1/2 of the declining phase, paralleled that in blood closely. An exception was T1/2 of the trans-4R,5R-isomer in the kidney, which was over twice as long as that in blood. Finally, the blood concentrations of an isomer correlated well with the concentrations in a given tissue [r = 0.73-0.99; p ≤ 0.004-0.001, depending on the isomer and tissue].

Blood and brain tissue samples from one to three rats per isomer were analyzed for the presence of the active 4-methylaminorex metabolites norephedrine and norpseudoephedrine. Norephedrine was found occasionally only in the samples taken from the rats treated with the cis-isomers, whereas norpseudoephedrine was likewise found only in the rats treated with the trans-isomers. However, the concentrations of these metabolites were almost always below the limit of quantification (0.01 μg/ml or μg/g). The measured concentrations were as follows: norephedrine in the brain 0.016 μg/g (30 min) after cis-4R,5S, and 0.035 μg/g (30 min) and 0.031 μg/g (60 min) after cis-4S,5R; norpseudoephedrine in blood 0.014 μg/g (120 min) and 0.011 μg/g (150 min) after trans-4S,5S; norpseudoephedrine in the brain 0.039 μg/g (30 min) after trans-4R,5R. Furthermore, concentrations below the quantification limit were observed as follows: norephedrine in blood (30 and 60 min) after cis-4S,5R-isomer; norpseudoephedrine in blood (30 min) after trans-4S,5S. Because the highest concentrations measured were generally lower than those observed after the administration of norephedrine or norpseudoephedrine at pharmacologically relevant doses (Frosch, 1977; Meyer and Portmann, 1982; Coutts et al., 1984; Henderson and Fuller, 1992; Kamakura and Satake, 1998), the samples from the rest of the rats were not analyzed.

Discussion

The present study describes differences between the stereoisomers of 4-methylaminorex in the pharmacokinetics and tissue distribution. The trans-4S,5S-, cis-4R,5S-, and cis-4S,5R-isomers behaved somewhat comparably, each demonstrating low oral bioavailability, but extensive distribution with good penetrability into the brain, and relatively short elimination half-life, whereas trans-4S,5S-isomer differed from the others, with high oral bioavailability and over 3-fold longer elimination half-life. The typical rank order of the isomers' tissue concentrations was kidney > liver > brain > muscle > fat ≈ blood. The elimination rates in these tissues usually paralleled those in blood. Finally, the metabolites norephedrine and norpseudoephedrine were detectable, but only at low concentrations, in blood and the brain.

Pharmacokinetics and tissue distribution properties are important factors determining patterns of drug consumption. High potential for harmful use with persistent self-administration can be predicted for a drug with high Fbioav, good penetrability to the brain, short T1/2, and high free drug CL (Quinn et al., 1997). This profile agrees with the kinetic properties of trans-4S,5S-, cis-4R,5S-, and cis-4S,5R-isomers administered intravenously, but not orally, as this route demonstrated low Fbioav. Indeed, the pharmacokinetic parameters of the three isomers calculated from the intravenous data correspond to those reported previously for amphetamine, methamphetamine, and methylenedioxymethamphetamine (Cho et al., 1990; Hutchaleelaha et al., 1994; Riviere et al., 2000). This parallel prompts speculation on whether the consumption patterns of these isomers would resemble abuse habits characteristic of highly addictive amphetamines.

Unlike the other isomers, trans-4R,5R-isomer demonstrated over 3-fold longer T1/2el and markedly better Fbioav after intraperitoneal and oral administrations. These findings confirm and expand upon the results of our earlier studies, in which the concentrations of trans-4R,5R-isomer seemed to be higher than those of the other isomers in rat brain microdialysis samples, or in rat serum and brain at a single time point (Kankaanpää et al., 2001, 2002). Thus, the rank order of blood and brain concentrations of the stereoisomers was trans-4R,5R-> cis-4R,5S-≈ cis-4S,5R-≈ trans-4S,5S-isomer. This is clearly incompatible with the rank order of potency trans-4S,5S-> cis-4R,5S-≈ cis-4S,5R-> trans-4R,5R-isomer observed in previous behavioral and neurochemical studies (Glennon and Misenheimer, 1989; Batsche et al., 1994; Ashby et al., 1995; Kankaanpää et al., 2002). The dissociation of in vivo drug effects from pharmacokinetics is further evidenced by the observation that trans-4R,5R-isomer administered intraperitoneally or subcutaneously induced behavioral activation more slowly than the other isomers (Glennon and Misenheimer, 1989; Batsche et al., 1994; Kankaanpää et al., 2002), although their kab values and T1/2ab values are equal, indicating a similar rate of absorption. Furthermore, we found no differences between the concentrations of the cis-isomers in blood, brain, or virtually any other tissue, despite the finding that they yielded qualitatively a different behavioral response at equipotent doses: locomotor activity suffused after cis-4S,5R-isomer, contrasted with stereotyped behavior after cis-4R,5S-isomer (Batsche et al., 1994). Thus, if our findings are considered jointly with the previous data, it seems that the differences between the stereoisomers in both potency and behavioral response profile are not due to pharmacokinetic factors.

There were marked differences in the distribution of the stereoisomers among the tissues. The highest concentrations with all the stereoisomers were found in the kidney, which is compatible with the previous suggestion that the 4-methylaminorex elimination occurs primarily via this organ, with roughly 60% of total excretion as unchanged drug (Henderson et al., 1995). The next highest concentrations were mostly found in the liver, except for cis-4R,5S-isomer, which showed very low liver concentrations, followed by brain, muscle, and then equal concentrations in fat and blood. In almost all the tissues, the elimination rate of the isomer paralleled rather closely that in blood, which indicates that no unexpected accumulation should occur in these tissues with repeated administration. An exception was the kidney concentration of trans-4R,5R-isomer, which fell over twice as slow as that in blood. As this isomer also showed the slowest elimination rate from blood and the other tissues, it would be tempting to speculate that trans-4R,5R-isomer may penetrate more poorly than the others into urine, thereby resulting in the slower elimination.

Little is currently known about the metabolic fate of 4-methylaminorex. After the administration of a mixture containing mainly the cis-isomers, three metabolites were recognized in rat urine: the synthetic precursor norephedrine and two pharmacologically poorly characterized oxazoline-derivatives, 5-phenyl-4-methyl-2-oxazolidinone and 2-amino-5-(p-hydroxyphenyl)-4-methyl-2-oxazoline (Henderson et al., 1995). It was not possible to analyze these oxazolines because of the lack of reference standards, but norephedrine was found as a metabolite in blood and the brain after the cis-isomers, as against norpseudoephedrine that was present only after the trans-isomers. Their concentrations, however, remained below the limit of quantification in most samples. Even when quantified, the concentrations seemed to be low; for instance, when administered at behaviorally relevant doses, norephedrine concentrations were at least 40-fold higher in rat brain and 20-fold higher in blood than in our study (Coutts et al., 1984; Henderson and Fuller, 1992; Kamakura and Satake, 1998). Although no such precise data on norpseudoephedrine are available, it seems unlikely that its effective concentrations are dozens of times smaller than those of norephedrine. Certainly concentrations that release dopamine from rat brain tissue in vitro (Kalix, 1983; Kalix et al., 1987), or concentrations considered therapeutic in human plasma after a single drug dose (Meyer and Portmann, 1982), are several times higher than the highest concentrations in our study. Together, it seems that the cis- and trans-isomers can be metabolized into norephedrine and norpseudoephedrine, respectively, but their contribution to 4-methylaminorex pharmacology is probably of minor significance.

It should be noted that the analytical method used in the present study distinguishes the cis- and trans-isomers by their retention times, but makes no distinction between the enantiomers (two cis- or two trans-isomers). The current method, however, was considered applicable to our purposes for two reasons. First, each stereoisomer was administered separately in our study, and second, no signs of cis-isomer were found after the administration of a trans-isomer, or vice versa, thereby ruling out the conversion of one isomer into another.

In summary, the present study describes the differences in pharmacokinetics and tissue distribution between the stereoisomers of 4-methylaminorex, a potential psychostimulant drug of abuse. The trans-4S,5S-, cis-4R,5S-, and cis-4S,5R-isomers behaved kinetically somewhat comparably, whereas trans-4R,5R-isomer differed from the others in having high oral Fbioav, longer T1/2el, and higher tissue concentrations. However, these differences are not compatible with, and thus may not account for, the distinct pharmacodynamic profiles of the isomers observed in previous research. Furthermore, although the metabolic turnover of the isomers to norephedrine and norpseudoephedrine was evident, due to the low concentrations this may not significantly contribute to 4-methylaminorex pharmacology. Together, the spatial structure of 4-methylaminorex seems to have a distinct impact on its kinetic, metabolic, and dynamic properties, and these need to be considered in pharmacological and analytical studies.

Meyer J and Portmann P (1982) Fluorometric determination of the d-norpseudoephedrine in the plasma. Plasma levels during the resorption from the free and extended-release form. Pharm Acta Helv57:12-15.